Extended Data Fig. 9: 40h dm-αKG treatment of blastoids leads to improved attachment and trophoblast lineage differentiation.
a, Schematic of blastoid attachment assays in IVC1-2 medium on IbiTreat µ-plates. b, Quantification of attachment efficiency 24 h after plating blastoids, treated with 40 h 0 mM or 4mM dm-αKG. Data shows the mean attachment percentages and data points of N = 7 biological replicates, n = 560. c, Left panel: representative IF images of NANOG, GATA3 and GATA6 in blastoids treated for 40 h with 0 or 4 mM of dm-αKG at d4 of attachment. White arrows indicate GATA6 single positive cells. Scale bar = 100 μm. Right panel: IF quantification. Data shows the mean percentages of structures with all 3 lineages (NANOG, GATA3 and GATA6) and data points of N = 4 biological replicates, n = 181. d, Schematic of blastoid attachment assays in N2B27 + E2 medium on IbiTreat µ-plates coated with ECM. e, Representative brightfield images of blastoids with or without 40 h 4mM dm-αKG treatment at d4 and d5 post attachment. Black arrows indicate extravillous trophoblast-like cells. Scale bar = 160 μm. f, Quantification of size (diameter) of blastoids treated for 40 h with 0 or 4 mM of dm-αKG at d4 and d5 of attachment. N = 4, n = 142 structures. Violin plot shows: center line: median; bottom and upper line: 25th and 75th percentiles. g, Representative IF images of DAPI, hCGB and HLA-G in blastoids treated for 40 h with 0 or 4 mM of dm-αKG at d7 of attachment. Scale bar = 100 μm. h, IF quantification of structures with syncytiotrophoblast- (SCT) and extravillous trophoblast- (EVT) like cells. Data shows the mean percentages of structures with hCGB or HLA-G positive cells and data points of N = 4 biological replicates, n = 115. P values were calculated by two-sided paired t-tests (c,h) or two-sided unpaired t-tests (b,f).
